Invertebrate lysozymes: Diversity and distribution, molecular mechanism and in vivo function

Lysozymes are antibacterial enzymes widely distributed among organisms. Within the animal kingdom, mainly three major lysozyme types occur. Chicken (c)-type lysozyme and goose (g)-type lysozyme are predominantly, but not exclusively, found in vertebrate animals, while the invertebrate (i)-type lysozyme is typical for invertebrate organisms, and hence its name. Since their discovery in 1975, numerous research articles report on the identification of i-type lysozymes in a variety of invertebrate phyla. This review describes the current knowledge on i-type lysozymes, outlining their distribution, molecular mechanism and in vivo function taking the representative from Venerupis philippinarum (formerly Tapes japonica) (Vp-ilys) as a model. In addition, invertebrate g-type and ch-type (chalaropsis) lysozymes, which have been described in molluscs and nematodes, respectively, are also briefly discussed.     

Cloning and expression analysis of c-type and g-type lysozymes in yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>)

Lysozymes have important roles in innate immune system. Here, a c-type and a g-type lysozyme were identified from yellow catfish (Pelteobagrus fulvidraco). The deduced amino acid sequences of both lysozymes were conserved in catalytic sites and structural features as compared to their counterparts from other species. It was interesting that the g-type lysozyme possessed a signal peptide. The c-type and g-type lysozymes had the highest identity 89.4 and 76.2 % with that from channel catfish respectively. Phylogenetic analysis showed that the two lysozymes had a closely relationship with that from channel catfish and Astyanax mexicanus. Lysozymes from one order could form more than one clade in the phylogenetic tree, which indicated the gene duplications in evolution. Expression analysis with real time quantitative PCR revealed that the two lysozyme genes were constitutively expressed in all the tested tissues. The highest expression of c-type lysozyme was observed in liver, followed by spleen, head kidney, and trunk kidney, while the g-type lysozyme had highest expression in intestine, followed by spleen, head kidney, and trunk kidney. The mRNA levels of both genes were all up-regulated after challenging with Aeromonas hydrophila. However, there were differences in tissues and time points when the mRNA levels reached its peak between the two lysozymes. It indicated the diversity in regulation mechanisms and detailed functions among lysozymes. Taking together, these results will benefit the understanding of yellow catfish lysozymes.     

Role of Lysozyme Inhibitors in the Virulence of Avian Pathogenic Escherichia coli

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors.     

A new lysozyme from the eastern oyster, <Emphasis Type="Italic">Crassostrea virginica</Emphasis>, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion

Background  

Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes.     

Lysozyme activity in animal extracts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Lysozyme activity was detected after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing 0.2% (W/V) autoclaved Micrococcus lysodeikticus cells as substrate. 2. Lysozyme activity appeared as clear lysis zones after incubation of opaque gels at 37 degrees C in buffered Triton X-100. 3. As low as 0.1 pg of purified hen egg white lysozyme could be detected after 16 hr incubation at pH 6.5. 4. Bands with lytic activity from kidney and pancreas acetone powders, bird's egg whites and vitelline membranes, animal sera and human saliva corresponded to c-type (Mr 14,500), g-type (Mr 20,500) or both lysozymes as far as molecular weight is concerned. 5. Some extracts, like porcine kidney, exhibited more than two bands. 6. Bands with lytic activity migrating at the level of g-type lysozymes were detected in some kidney and pancreas extracts.     

Lysozyme activity of the salivary gland secretion of the medicinal leeches <Emphasis Type="Italic">H. verbana,H. medicinalis</Emphasis>, and <Emphasis Type="Italic">H. orientalis</Emphasis>

Salivary gland secretions of three species of the medicinal leech differ in the level of their lysozyme, and peptidoglycan-lysing activity. Using a synthetic fluorogenic substrate, 4-methylumbelliferyltetra N-acetyl-β-chitotetraoxide, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of these species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is suggested that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize these substrates including lysozymes other than i-type (invertebrate) lysozymes.     

Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus

Lysozyme gene from Fenneropenaeus indicus was cloned, expressed in Escherichia coli and characterized. The cDNA consists of 477 base pairs and encodes amino acid sequence of 159 residues. F. indicus lysozyme had high identity (98 %) with Fenneropenaeus merguiensis and Fenneropenaeus chinensis and exhibits low to moderate identities with lysozymes of other invertebrates and vertebrates. This lysozyme is presumed to be chicken types as it possesses two catalytic and eight cysteine residues that are conserved across c-type lysozymes and a c-terminal extension, which is a characteristic of lysozymes from marine invertebrates. Further, the antimicrobial properties of the recombinant lysozyme from F. indicus were determined in comparison with recombinant hen egg white lysozyme. This exhibited high activity against a Gram-negative pathogenic bacterium Salmonella typhimurium and two fungal strains Pichia pastoris and Saccharomyces cerevisiae in turbidimetric assay. Distribution of lysozyme gene and protein in tissues of shrimps infected with white spot syndrome virus revealed that the high levels of lysozyme are correlated with low and high viral load in abdominal muscle and tail, respectively. In conclusion, lysozyme from F. indicus has a broad spectrum of antimicrobial properties, which once again emphasizes its role in shrimp innate immune response.     

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein–protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the β domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein–protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.     

The activity parameters,cDNA cloning and mRNA expression of lysozyme in Cyclina sinensis (Bivalvia,Veneridae) responding to the pathogenic bacterium Vibrio anguillarum

ABSTRACT

Lysozyme is an important component of the innate immune response against pathogen infection. The functional forms have been well-studied in the c-type lysozymes of vertebrates but less so in i-type lysozymes prevalent in most invertebrate animals. Cyclina sinensis is a commercially important marine venerid bivalve that is abundant and widely distributed around the maritime coasts of Asia. We obtained an i-type lysozyme (i-lyz) gene in C. sinensis by large scale EST sequencing of a SMART-cDNA library. In C. sinensis, the i-lyz gene encodes 181 amino acids. The lysozyme activities and the mRNA levels of the i-lyz gene in haemocytes were upregulated during the infection by a bacterium, Vibrio anguillarum. The lysozyme activity in haemocytes was increased after 12?h infection by V. anguillarum, and reached the highest level at 24?h post-infection, which was significantly higher than that of the control group (P?<?0.01). The expression level of the i-lyz gene in haemocytes was increased after 3?h infection of Vibrio, and reached the highest level at 6?h post-infection. A recombinant i-lyz was obtained through prokaryotic expression which, when isolated and purified, had a molecular weight of approximately 19?kDa. Western blotting was used to validate the expression of the fusion protein. Furthermore, we demonstrate that the recombinant i-lyz protein has obvious bacteriostatic activity upon Escherichia coli whereby the K value of the curve was significantly lower than control and blank groups. Our study shows that the C. sinensis i-type lysozyme plays an important role in the immune defence against V . anguillarum and provides a theoretical reference for clarifying the mechanism of immune response against pathogen invasion in this bivalve.     

The gene of chlamysin, a marine invertebrate-type lysozyme, is organized similar to vertebrate but different from invertebrate chicken-type lysozyme genes

In a recent publication we reported the protein purification, characterization, and the gene isolation of a cDNA encoding the antibacterial cold-active lysozyme-like protein chlamysin from the marine bivalve Chlamys islandica. A 4.2 kb genomic chlamysin gene has now been amplified and sequence-analyzed. By comparison to the cDNA sequence and its translation product, the coding region was found separated in four exons of 38-252 bp. The introns range in size from 0.8 to 1.5 kb, and have traditional spliceosomal intron 5'-GT donor and 3'-AG acceptor sites for splicing. Two of the introns contain multiple copies of three sequence motifs not found repeated in other published genes. The over-all gene organization of chlamysin resembles chicken-type (c-type) lysozyme genes in vertebrates, but is different from the three-exon structure in invertebrate c-type lysozyme genes. A phylogenetic analysis of invertebrate-type (i-type) and c-type lysozyme proteins demonstrated a large evolutionary distance between the i-type and the c-type enzyme classes. Exons of the i-type genes are not equally organized according to their homolog protein domains.     

An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity

Lysozymes function in animal immunity. Three types of lysozyme have been identified in animal kingdom and most lysozymes identified from bivalve molluscs belong to the invertebrate (i) type. In this research, we cloned and sequenced a new i-type lysozyme, named MmeLys, from the Asiatic hard clam Meretrix meretrix. MmeLys cDNA was constituted of 552 bp, with a 441 bp open reading frame encoding a 146 amino acid polypeptide. The encoded polypeptide was predicted to have a 15 amino acid signal peptide, and a 131 amino acid mature protein with a theoretical mass of 14601.44 Da and an isoelectric point (pI) of 7.14. MmeLys amino acid sequence bore 64% identity with the Manila clam (Venerupis philippinarum) i-type lysozyme and was grouped with other veneroid i-type lysozymes in a bivalve lysozyme phylogenetic tree predicted using Neighbor-Jointing method. Recombinantly expressed MmeLys showed lysozyme activity and strong antibacterial activity against Gram positive and Gram negative bacteria. MmeLys mRNA and protein were detected to be mainly produced in hepatopancreas and gill by the methods of semi-quantitative RT-PCR and western blotting. In addition, MmeLys gene expression increased following Vibrio parahaemolyticus challenge. Results of this research indicated that MmeLys represents a new i-type lysozyme that likely functions in M. meretrix immunity.     

Phylogenetic Analysis of Invertebrate Lysozymes and the Evolution of Lysozyme Function

We isolated and sequenced the cDNAs coding for lysozymes of six bivalve species. Alignment and phylogenetic analysis showed that, together with recently described bivalve lysozymes, the leech destabilase, and a number of putative proteins from extensive genomic and cDNA analyses, they belong to the invertebrate type of lysozymes (i type), first described by Jollès and Jollès (1975). We determined the genomic structure of the gene encoding the lysozyme of Mytilus edulis, the common mussel. We provide evidence that the central exon of this gene is homologous to the second exon of the chicken lysozyme gene, belonging to the c type. We propose that the origin of this domain can be traced back in evolution to the origin of bilaterian animals. Phylogenetic analysis suggests that i-type proteins form a monophyletic family. Received: 21 May 2001 / Accepted: 22 October 2001     

Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica)

Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.     

Biochemical characterization of lysozymes present in egg white of selected species of anatid birds

The isolation of lysozyme from the egg white of several representative species of waterfowl is described. The purified lysozymes were analyzed to determine the type and molecular weight of each enzyme. All enzymes found in duck egg whites were found to be of the c-type. In contrast all true geese, and the mute swan species as well as the northern blackneck screamer contain lysozyme g in their egg white.